NK-2 Homeobox Gene. A rabbit antibody was obtained that is directed against Drosophila NK-2 homeodomain protein. The earliest expression of NK-2 protein detected was in stage 6 embryos, approximately 1 hour after the initial appearance of NK-2 mRNA. NK-2 protein was found in 2 medial clusters of neuroblasts per hemisegment, which confirms part of the pattern found for NK-2 mRNA. Recently, other investigators have shown that the NK-2 gene and the ventral nervous system defective gene (vnd) are the same gene. Additional reports show that vnd activates proneural genes of the achaete-scute complex of genes (achaete and lethal of scute), which in turn activate proepidermal genes in the Enhancer of split (E(spl)) complex of genes. These results suggest that NK-2 protein is required for commitment of some cells to the neural pathway of development. We find that mutation of Delta or deletion of the E(spl) complex of genes results in overproduction of NK-2 positive thoracic neuroblasts and aberrant patterns of NK-2 positive neuroblasts and neurons. These results suggest that lateral inhibition may involve repression of the NK-2 gene by helix-loop-helix proteins encoded by genes in the E(spl) complex. The primary specificity determinants of high affinity NK-2 homeodomain binding to DNA were shown to be interactions between Ile-47 of the NK-2 homeodomain and A3 and A4 residues in the (+) strand of DNA and between Tyr-54 and T-4, C5, and A6 in the (-) strand of DNA. Mutagenesis of DNA encoding the homeodomain of NK-2 resulting in the replacement of the codon for Tyr-54 by a codon for Met-54 reduced the affinity of the mutated NK-2 homeodomain for the consensus NK-2 binding site in DNA.